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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2012 - April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
adopted 1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-3-{4-[2-hydroxy-3-(prop-2-enoyloxy)propoxy]butoxy}propyl prop-2-enoate
EC Number:
701-230-0
Cas Number:
52408-42-1
Molecular formula:
C16H26O8
IUPAC Name:
2-hydroxy-3-{4-[2-hydroxy-3-(prop-2-enoyloxy)propoxy]butoxy}propyl prop-2-enoate
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weekds
- Weight at study initiation: on average 27.9g
- Assigned to test groups randomly: yes
- Housing: single in Makrolon type M II cages
- Diet (e.g. ad libitum): standardized pelleted feed ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (4mL/kg b.w.) + corn oil
- Justification for choice of solvent/vehicle: solubility of the test substance, historic control data is available
- Concentration of test material in vehicle: 25 - 100mg/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved by thourough shaking in DMSO (4 mL/kg) and than emulsified in corn oil (up to 10 mL/kg). All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
one admistration
Frequency of treatment:
one admistration
Post exposure period:
24h , 48h (only high dose)
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP), Vincristine sulfate (VCR)
- Justification for choice of positive control(s): both substances are well-established reference clastogens and aneugens, respectively
- Route of administration: via gavage in deionized water
- Doses / concentrations: 20mg/kg (CPP), 0.15mg/kg (VCR)

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest to determine the acute oral toxicity in males and females, lethality was observed at the recommended highest dose of 2 000 mg/kg body weight. In addition, clear signs of toxicity were observed: piloerection, hunched posture and reduced general condition. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals were used in the main experiment. Based on the data of the pretest a dose of 1 000 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
One drop of isolated bone marrow cells in FCS was dropped onto a clean microscopic slide, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained with eosin and methylene blue (modified May-Grünwald solution) for about 5 minutes. After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes. Subsequently, the slides were stained with Giemsa solution for about 15 minutes. After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Fraction of polychromatic erythrocytes containing micronuclei (index of clastogenic or aneugenic activity)
• Fraction of normochromatic erythrocytes containing micronuclei (24h value: control value for situation before test substance administration)
• Ratio of polychromatic to normochromatic erythrocytes (indicator that the test substance reached the bone marrow)
• Number of small micronuclei (d < D/4) and of large micronuclei (d = D/4) [d = diameter of micronucleus, D = cell diameter] (differentiation between a clastogenic and spindle poison effect, respectively)
Evaluation criteria:
Acceptance criteria
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. = 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

A finding is considered positive, if the number of PCEs containing micronuclei is statistically significant and dose-related increased, and the number of PCEs containing micronuclei exceeds both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative, if the number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PCE to NCE ratio suggests a slight inhibition of erythropoiesis at 1000mg/kg after 48h
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Negative and positive controls :
Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. The single oral administration of the vehicle DMSO/corn oil in a volume of 10 mL/kg body weight led to 0.9‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8‰ after the 48-hour sacrifice interval, respectively.

Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (16.4‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a statistically significant increase (24.8‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 8.5‰ was attributable to large micronuclei.


Results with the test susbtance:
After the single administration of the highest dose of 1 000 mg/kg body weight, 0.9‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 0.8‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of 1.1‰ (500 mg/kg group) and 0.7‰ (250 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.
A slight inhibition of erythropoiesis induced by the treatment of mice with test substance was detected at 1 000 mg/kg body weight at 48 hours sacrifice interval. The ratio of polychromatic to normochromatic erythrocytes was influenced compared to the respective vehicle control group which is an indication of target organ toxicity.

Clinical signs :
The administration of the test substance did not lead to clinical signs of toxicity in the main experiment. However, lethality and clear clinical observations were found in the pretest at a two-fold higher dose of 2 000 mg/kg body weight.

Applicant's summary and conclusion

Conclusions:
Under the condition of this study, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.
Executive summary:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to 5 male animals per group at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours (all dosages and control), and 48 hours (high dose and vehicle control) after administration. The preparations were stained, and 2 000 polychromatic erythrocytes were evaluated per animal. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

According to the results of the present study, there are thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (250 mg/kg, 500 mg/kg and 1 000 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range. Nor were large micronuclei (d = D/4) observed either in the vehicle control group or in the three dose groups treated with test substance.

Based on the pretest, 1 000 mg/kg body weight was defined as maximum tolerated dose (MTD) due to lethality observed at 2 000 mg/kg body weight. Although no signs of toxicity were found after treatment with 1 000 mg/kg body weight in the main experiment, bioavailability of the test substance in the target organ was shown by reduction of polychromatic erythrocytes at 48-hour sacrifice interval.

In this study, after single oral administration of the vehicle DMSO/corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected.

Thus, under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.